Immunosenescence and vaccine efficacy revealed by immunometabolic analysis of SARS-CoV-2-specific cells in multiple sclerosis patients

Disease-modifying therapies (DMT) administered to patients with multiple sclerosis (MS) can influence immune responses to SARS-CoV-2 and vaccine efficacy. However, data on the detailed phenotypic, functional and metabolic characteristics of antigen (Ag)-specific cells following the third dose of mRNA vaccine remain scarce. Here, using flow cytometry and 45-parameter mass cytometry, we broadly investigate the phenotype, function and the single-cell metabolic profile of SARS-CoV-2-specific T and B cells up to 8 months after the third dose of mRNA vaccine in a cohort of 94 patients with MS treated with different DMT, including cladribine, dimethyl fumarate, fingolimod, interferon, natalizumab, teriflunomide, rituximab or ocrelizumab. Almost all patients display functional immune response to SARS-CoV-2. Different metabolic profiles characterize antigen-specific-T and -B cell response in fingolimod- and natalizumab-treated patients, whose immune response differs from all the other MS treatments.


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Life sciences study design
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Sample size
Attune NxT software v.4.2.0 CyTOF software ver.10.7.1014 The original contributions presented in the study are included in the article/Supplementary Material/Source Data File.Further inquiries can be directed to the corresponding author.
Demographic and clinical characteristics of patients are reported in table 1. Sex was indicated.We do not have any information regarding gender.
We enrolled patients who were admitted to routine visit in the Nerological Center in the clinic whith the following inclusion and exclusion criteria.Patients were eligible for inclusion if they met the following criteria: a) a confirmed diagnosis of Relapsing-Remitting Multiple Sclerosis (RRMS), and b) a history of treatment with FTY, dimethyl fumarate, natalizumab, or teriflunomide for a minimum of six months, or having undergone at least two infusional cycles with rituximab/ocrelizumab or completed at least one full cycle of cladribine.Patients on ocrelizumab or rituximab, as per routine clinical practice, underwent SARS-CoV-2 vaccination at least six weeks before subsequent infusion or at least three months after the last infusion.Exclusion criteria comprised treatment with steroids during the preceding six weeks and a history of COVID-19 before vaccination.
The study was reviewed and approved by each participant, including healthy donors, provided informed consent according to Helsinki Declaration, and all uses of human material have been approved by the local Ethical Committee (Comitato Etico dell 'Area Vasta Emilia Nord, protocol number 199/ 2022, May 24th, 2020) and by the University Hospital Committee (Direzione Sanitaria dell'Azienda Ospedaliero Universitaria di Modena, protocol number 5974, February 24th, 2023).The patients/participants provided their written informed consent to participate in this study.
Due to the situation, i.e., the Covid pandemics, no sample size calculation was performed.However, according to our previous and large experience on the analysis of T cells, a sample size of 12 patients and 12 controls for cell phenotype was considered sufficient to detect relevant differences.However, for analysis of T cell phenotype, and function we were able to include 94 patients and 13 healthy

Antibodies
Antibodies used donors.Notwithstanding the fact that for some treatment groups we did not reach a numerosity of 12, due to the striking differences in the mechanisms of action of the drugs, we nevertheless also detected significant differences involving treatment groups with <12 patients per group, as reported in the paper, with regard to either antigen-specific T/B cell responses or metabolic features.
No data were excluded from the analysis.
No experimental replication was conducted in this study, as all samples were derived from primary human participants.However the reproducibility of the analysis was tested analysing the same samples in different days.Flow cytometers were aligned every day with QC beads.
Patients were not randomized.They were enrolled consecutively throughout the enrolment period, if inclusion/exclusion criteria were met, during routine clinical visits at the MS centre.Samples were then allocated to the different groups based on the disease-modifying treatment they were on.The differences in numerosity throughout treatment groups reflect the different frequencies of treatments prescribed per standard clinical practice at our center.Randomization was not carried out since we were not expecting the different treatment groups to be similar in relation to disease or immune cell status.We were, in fact, expecting a different effect of the Sars-CoV2-vaccine as a consequence of the differences between the groups and, in particular, of the different mechanisms of action of the prescribed disease-modifying treatments For obvious reasons, all blood from human beings is considered infected and treated as such, so fersearchers used the same procedures for all samples.So, samples were coded in the Multiple Sclerosis Clinic and taken to the lab, where blood was treated, stored and subsequently analyzed in a blind manner.Moreover, we applied unsupervised statistical analysis to avoid any possible influence of the operator.The senior authors of the paper (Cossarizza) did not open the key until the end of the study.
All the antibodies used are reported in supplementary tables 1-5, divided per panel.For each antibody, marker, label, clone, brand cat, lot and titer have been reported.Antibodies used for AIM assay (supplementary table 1).We used only monoclonal antibodies that are commercially available and have been validated by different companies.We have titrated each of them for the optimal use by flow cytometry, as recommended by the most recent guidelines for the use of cytometry in immunological studies (Cossarizza et al., Eur. J. Immunol. 2021: Hartmann et al Nat Biotecnology 2022).For more information about antibody validation by different brand visit www.biolegend.com; www.standardbiotools.com; www.bdbiosciences.com; www.abcam.com; www.novus.com; www.cellsignalling.com; www.thermofisher.com; www.beckmancoulter.comThe axis labels state the marker and fluorochrome used (e.g.CD4-FITC).
The axis scales are clearly visible.Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).
All plots are contour plots with outliers or pseudocolor plots.
A numerical value for number of cells or percentage (with statistics) is provided.

Sample preparation
Blood collection and isolation of mononuclear cells Up to 30 mL of blood were collected from each patient in vacuettes containing ethylenediamine-tetraacetic acid (EDTA).Blood was immediately processed.Isolation of peripheral blood mononuclear cells (PBMC) was performed using ficollhypaque according to standard procedures.For all experiments, except those related to metabolic investigation, PBMC were stored in liquid nitrogen in fetal bovine serum (FBS) supplemented with 10% dimethyl sulfoxide (DMSO).For metabolic investigation, PBMC were used immediately after isolation.Isolated PBMCs were thawed and rested for 6 hours.After resting, CD40-blocking antibody (0.5 mg/ml final concentration) (Miltenyi Biotec, Bergisch Gladbach, Germany) was added to the cultures 15 min before stimulation.PBMCs were cultured in 96-well plate in the presence of 15-mer peptides with 11-amino acids overlap, covering the complete sequence of Wuhan SARS-CoV-2 Spike glycoprotein (PepTivator SARS-CoV-2 Prot_S complete, Miltenyi Biotec, Bergisch Gladbach, Germany) together with 1 g/mL of anti-CD28 (Miltenyi Biotec, Germany).PBMCs were stimulated for 18 h at 37 °C in a 5% CO2 atmosphere in complete culture medium (RPMI 1640 supplemented with 10% fetal bovine serum and 1% each of Lglutamine, sodium pyruvate, nonessential amino acids, antibiotics, 0.1M HEPES, 55M -mercaptoethanol).For each stimulated sample, an unstimulated one was prepared, as negative control.After stimulation, cells were washed with PBS and stained with PromoFluor IR-840 (Promokine, PromoCell, Heidelberg, Germany) for 20 minutes at room temperature (RT).
Detection of SARS-CoV-2-specific B cells Thawed PBMC were washed twice with RPMI 1640 supplemented with 10% fetal bovine serum and 1% each of L-glutamine, sodium pyruvate, nonessential amino acids, anti-biotics, 0.1M HEPES, 55M -mercaptoethanol and 0.02 mg/ml DNAse.PBMC were washed with PBS and stained using viability marker PromoFluor IR-840 (Promokine, PromoCell, Heidelberg, Germany) for 20 min at RT in PBS.Next, cells were washed with PBS and stained for 15 min at RT with streptavidin-AF700 (decoy channel; ThermoFisher Scientific, USA) to remove false positive SARS-CoV-2-specific B cells.After washing with FACS buffer, cells were stained with biotinylated full-length SARS-CoV-2 spike protein (R&D Systems, Minneapolis) labelled with different streptavidin-fluorophore conjugates.Full-length biotinylated spike protein was mixed and incubated with streptavidin-BUV661(Becton Dickinson) or streptavidin-BV650 (BioLegend) at a 6:1 mass ratio for 15min at RT.All samples were stained with both biotinylated streptavidin for 1h at 4°C.Then, cells were washed with FACS buffer and stained for 20 min at RT with DuraClone IM B cells (Beckman Coulter, Brea, CA) containing the following lyophilized directly conjugated mAbs: anti-IgD-FITC, CD21-PE, CD19-ECD, CD27-PC7, CD24-APC, CD38-AF750, anti-IgM-PB, CD45-KrO to which following drop-in antibodies were added: CD71-BUV395, CD20-BV785, anti-IgG-BUV496 and anti-IgA-PerCP-Vio700.Samples were acquired on a CytoFLEX LX flow cytometer (Beckman Coulter).A minimum of 1,000,000 cells per sample were acquired.All reagents used for B cell phenotype are reported in Supplementary Table 3.All mAbs added to DuraClone IM B cells were previously titrated on human PBMCs and used at the concentration giving the best signal-to-noise ratio.The gating strategy used to identify Ag and Ag+ B cells is reported in the Supplementary Figure 11.

Table 5
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